Background: The nematode Caenorhabditis elegans is being assessed as an alternative model organism as part of an interagency effort to develop better means to test potentially toxic substances. As part of this effort, assays that use the COPAS Biosort flow sorting technology to record optical measurements (time of flight (TOF) and extinction (EXT)) of individual nematodes under various chemical exposure conditions are being developed. A mathematical model has been created that uses Biosort data to quantitatively and qualitatively describe C. elegans growth, and link changes in growth rates to biological events. Chlorpyrifos, an organophosphate pesticide known to cause developmental delays and malformations in mammals, was used as a model toxicant to test the applicability of the growth model for in vivo toxicological testing.

Methodology/principal findings: L1 larval nematodes were exposed to a range of sub-lethal chlorpyrifos concentrations (0-75 microM) and measured every 12 h. In the absence of toxicant, C. elegans matured from L1s to gravid adults by 60 h. A mathematical model was used to estimate nematode size distributions at various times. Mathematical modeling of the distributions allowed the number of measured nematodes and log(EXT) and log(TOF) growth rates to be estimated. The model revealed three distinct growth phases. The points at which estimated growth rates changed (change points) were constant across the ten chlorpyrifos concentrations. Concentration response curves with respect to several model-estimated quantities (numbers of measured nematodes, mean log(TOF) and log(EXT), growth rates, and time to reach change points) showed a significant decrease in C. elegans growth with increasing chlorpyrifos concentration.

Conclusions: Effects of chlorpyrifos on C. elegans growth and development were mathematically modeled. Statistical tests confirmed a significant concentration effect on several model endpoints. This confirmed that chlorpyrifos affects C. elegans development in a concentration dependent manner. The most noticeable effect on growth occurred during early larval stages: L2 and L3. This study supports the utility of the C. elegans growth assay and mathematical modeling in determining the effects of potentially toxic substances in an alternative model organism using high-throughput technologies.

pone-0007024-g005: Growth rates of C. elegans after chlorpyrifos exposure.Estimated growth rates of log(EXT) per h (left panel) and log(TOF) per h (right panel) as functions of chlorpyrifos concentration. Growth rates are shown for three sections: initial growth rates before the first change point (blue), growth rates between change points (red), and growth rates after the second change point (green). Solid lines correspond to negative exponential functions with a common lower asymptote fit to the estimated growth rates. Nematodes exposed to chlorpyrifos concentrations greater than 30 µM did not grow to the third section.

Mentions:
To analyze the effects of chlorpyrifos on the estimated growth rates, a negative exponential function, , was fit to the growth rates in each of the three sections as functions of concentration (Fig. 5; equations for each section are presented in Supporting Information File S5). The strength of the chlorpyrifos effect on the growth rate is indicated by B. The final models for the three sections were chosen using the Akaike information criterion [22]. For every section, B was significantly greater than zero, indicating a significant inhibitory effect of chlorpyrifos on growth. In addition, the effect increased with increasing chlorpyrifos concentration for each of the three sections, further indicating concentration dependent growth inhibition (Fig. 5; Table 2). The functions eventually reached the same minimum growth rate for all three sections (Fig. 5), which may represent the minimum growth rate that the model could detect.

pone-0007024-g005: Growth rates of C. elegans after chlorpyrifos exposure.Estimated growth rates of log(EXT) per h (left panel) and log(TOF) per h (right panel) as functions of chlorpyrifos concentration. Growth rates are shown for three sections: initial growth rates before the first change point (blue), growth rates between change points (red), and growth rates after the second change point (green). Solid lines correspond to negative exponential functions with a common lower asymptote fit to the estimated growth rates. Nematodes exposed to chlorpyrifos concentrations greater than 30 µM did not grow to the third section.

Mentions:
To analyze the effects of chlorpyrifos on the estimated growth rates, a negative exponential function, , was fit to the growth rates in each of the three sections as functions of concentration (Fig. 5; equations for each section are presented in Supporting Information File S5). The strength of the chlorpyrifos effect on the growth rate is indicated by B. The final models for the three sections were chosen using the Akaike information criterion [22]. For every section, B was significantly greater than zero, indicating a significant inhibitory effect of chlorpyrifos on growth. In addition, the effect increased with increasing chlorpyrifos concentration for each of the three sections, further indicating concentration dependent growth inhibition (Fig. 5; Table 2). The functions eventually reached the same minimum growth rate for all three sections (Fig. 5), which may represent the minimum growth rate that the model could detect.

Bottom Line:
Concentration response curves with respect to several model-estimated quantities (numbers of measured nematodes, mean log(TOF) and log(EXT), growth rates, and time to reach change points) showed a significant decrease in C. elegans growth with increasing chlorpyrifos concentration.Statistical tests confirmed a significant concentration effect on several model endpoints.The most noticeable effect on growth occurred during early larval stages: L2 and L3.

Background: The nematode Caenorhabditis elegans is being assessed as an alternative model organism as part of an interagency effort to develop better means to test potentially toxic substances. As part of this effort, assays that use the COPAS Biosort flow sorting technology to record optical measurements (time of flight (TOF) and extinction (EXT)) of individual nematodes under various chemical exposure conditions are being developed. A mathematical model has been created that uses Biosort data to quantitatively and qualitatively describe C. elegans growth, and link changes in growth rates to biological events. Chlorpyrifos, an organophosphate pesticide known to cause developmental delays and malformations in mammals, was used as a model toxicant to test the applicability of the growth model for in vivo toxicological testing.

Methodology/principal findings: L1 larval nematodes were exposed to a range of sub-lethal chlorpyrifos concentrations (0-75 microM) and measured every 12 h. In the absence of toxicant, C. elegans matured from L1s to gravid adults by 60 h. A mathematical model was used to estimate nematode size distributions at various times. Mathematical modeling of the distributions allowed the number of measured nematodes and log(EXT) and log(TOF) growth rates to be estimated. The model revealed three distinct growth phases. The points at which estimated growth rates changed (change points) were constant across the ten chlorpyrifos concentrations. Concentration response curves with respect to several model-estimated quantities (numbers of measured nematodes, mean log(TOF) and log(EXT), growth rates, and time to reach change points) showed a significant decrease in C. elegans growth with increasing chlorpyrifos concentration.

Conclusions: Effects of chlorpyrifos on C. elegans growth and development were mathematically modeled. Statistical tests confirmed a significant concentration effect on several model endpoints. This confirmed that chlorpyrifos affects C. elegans development in a concentration dependent manner. The most noticeable effect on growth occurred during early larval stages: L2 and L3. This study supports the utility of the C. elegans growth assay and mathematical modeling in determining the effects of potentially toxic substances in an alternative model organism using high-throughput technologies.